The results indicated that C6- and C7-isoprenols have comparable RON boosting effects to isopentenols, making these two chemicals potential blendstocks for gasoline blends. We previously constructed the homoterpene biosynthesis platform as a proof of concept that introduces terpene structural diversity at the precursor stage. Here we further optimized this platform towards practical application. The most significant change is the upstream pathway to the key intermediate, 3- ketovaleryl-CoA. Like the natural LMVA pathways, our previous pathway starts from propionyl-CoA, condensed by a thiolase into 3-ketovaleryl-CoA. Two points led us to consider an alternative pathway to 3-ketovaleryl-CoA. First, thermodynamic analysis indicated the condensation reaction catalyzed by thiolase is endergonic with a positive Gibbs free energy change , suggesting the thiolase catalyzed condensation reaction is thermodynamically unfavorable. This calculation is consistent with the finding that PhaA homologs catalyze the degradation reaction better than the condensation reaction . Second, to our knowledge, almost all the reported thiolases that convert propionyl-CoA and acetyl-CoA into 3-ketovaleryl-CoA also convert two molecules of acetyl-CoA into acetoacetyl-CoA. Our later experiment using butyric acid as substrate in the beta-oxidation LMVA pathway suggests the LMVA pathway readily accepts acetoacetyl-CoA into IPP . Considering these two points and the relatively high concentration of acetyl-CoA in E. coli , we reasoned that it would be difficult for the thiolase LMVA pathway to make HIPP over IPP, complicating C16, C17 and C18 terpene biosynthesis. Instead, the beta-oxidation pathway is a more specific way to produce isopentenyl pyrophosphate analogs. Although background IPP production remains, either via the native MEP pathway or from endogenous acetoacetyl-CoA transformed by the LMVA pathway,curing marijuana the ratio of HIPP/IPP production is significantly improved, as the molar ratio of C6-isoprenol:isoprenol is over 60 in the production run using the E. coli BL21 ΔatoB host with 1 g/L valeric acid feeding.

The high production of HIPP and its dominant content in the isopentenyl pyrophosphate analog pool will benefit future homoterpene biosynthesis efforts. Using isoprenol analogs as the final product, we successfully optimized the flux to HIPP in the homoterpene biosynthesis platform. The enzymes after the LMVA pathway leading to complex terpenes are more challenging to optimize because of their elusive enzymology and unusual substrates. Following HIPP production, an IDI is supposed to isomerize HIPP to HDMAPP. We could not detect the corresponding alcohol of HDMAPP, C6-prenol, in the E. coli production run with the expression of NudB and the thiolase LMVA pathway containing the IDI from Bombyx mori. While this IDI was confirmed in vitro to transform HIPP to HDMAPP specifically , the absence of C6-prenol in the production run suggests that this IDI does not work well in E. coli, or the hydrolyzed product of HDMAPP, HDMAP, is not well accepted by the E. coli endogenous phosphatases. Incorporating this IDI into the optimized beta-oxidation LMVA pathway may increase HDMAPP production by increasing substrate supply. Other Lepidopteran IDIs are also candidates for enzyme screening of this step. At the same time, it is noteworthy that the regiospecificity of some Lepidopteran IDIs are low, because they transform HIPP to not only HDMAPP but also the -isomer of HDMAPP and isomers with a γ-δ double bond . After the isomerization, ideally, one molecule of HDMAPP is supposed to condense with different molecules of HIPP into homo-GPP , homo-FPP , and homoGGPP . To our knowledge, all the characterized FPPSs that produce homo-FPP also produce FPP, with varied substrate preferences. This substrate promiscuity could explain why our previous work only produced C16 homosesquiterpenes as low, or non-existent HDMAPP levels may hamper homo-FPP analog production. The overwhelming production of HIPP to IPP in the optimized platform here may increase the HIPP incorporation to produce more C16, C17, and even C18 FPPs. For other prenyl diphosphates, reported point mutations in prenyltransferases that change the product profiles could be applied on the lepidopteran FPPS to produce homo-GPP . Also, the structural basis of substrate preference for HIPP/HDMAPP derived prenyl diphosphates has been analyzed, the results of which are proposed to direct the engineering of non-lepidopteran prenyltransferases to accept HIPP/HDMAPP . Finally, terpene synthases cyclized the homo prenyl pyrophosphates to terpene scaffolds.

This step is the most challenging due to the lack of natural enzymes using homo prenyl pyrophosphates as the substrates. Future studies will focus on using rational design and directed evolution to alter the substrate specificity of canonical terpene synthases. Introducing extra carbon in the terpenes can significantly change their properties, exemplified by the optimized fuel properties of isoprenol biofuels. With comparable RON boosting effects and energy densities to isopentenols, C6- and C7-isoprenols have decreased water solubilities, making them better ingredients for fuel blends . In particular, C6-isoprenol derives fromvaleric acid, a key intermediate in the valerate biofuel platform . Numerous chemical reactions/processes have been developed to transform lignocellulose to valeric acid via levulinic acid , making our pathway promising to produce C6- isoprenol as a next-generation biofuel. Besides the simple terpenes we produce here, another example is -germacrene D, whose analog with two extra carbons, -14,15-dimethylgermacrene D shows a reversal in insect behavioral activity . Addressing those challenges in the homoterpene biosynthesis will enable the efficient production of various terpene analogs, leading to more diversified structures in the chemical portfolio for downstream applications. Proper adherence to combination antiretroviral medication therapy is critical for improving health outcomes in HIV . However, it is estimated that less than 40% of HIV+ individuals are retained in long-term healthcare management and only about one-quarter are virally suppressed in the U.S. . Further, approximately half of individuals prescribed antiretroviral medications do not fully adhere to their regimen . Suboptimal cART adherence is associated with increased viremia, immune suppression, increased risk of HIV transmission, and mortality . Numerous factors appear to be contributing to suboptimal cART adherence, including psychiatric comorbidities , lack of social support, severity of antiretroviral side effects, beliefs about self-efficacy and neurocognitive dysfunction .

With regard to the latter, HIV-associated neurocognitive deficits and cART non-adherence appear to have a cyclical relationship; that is, neurocognitive deficits can interfere with medication taking behaviors , which in turn can accelerate disease progression and further impair neurocognitive functioning . The historic complexity of combination antiretroviral therapies may also impose an undue burden on cognitive abilities necessary for adherence . The medications comprising a cART regimen may involve differing dosages and/or administration schedules, which could increase the risk of errors that negatively impact overall adherence, particularly among individuals with neurocognitive difficulties. Although the pill burden of cART is on a downward trajectory, polypharmacy remains common and these challenges are important given that cART is the gold standard for HIV treatment in the United States. Additionally, the prevalence of HIVassociated neurocognitive disorders may have even risen slightly in the cART era, with approximately 50% of the population affected . The elevated rates of HAND and domain-based impairment may be due to increasing lifespans resultant from high cART efficacy,dry racks for weed leading to longer exposure to both the presence of virus as well as potentially toxic long-term effects of the drug regimens, combined with comorbid processes such as cognitive aging . While HIV-associated neurocognitive deficits can be observed in a variety of neurocognitive domains, the prevalence of impairment in higher-order neurocognitive abilities, such as episodic memory in particular, are higher . In this study we therefore focus our attention on the role of episodic memory deficits in cART non-adherence. HIV-associated neural injury in frontal and temporal systems affect multiple aspects of episodic memory , including traditional retrospective memory for word lists, designs, and passages, as well as prospective, source, and temporal order memory . The profile of HIV-associated episodic memory deficits is heterogeneous , with prior studies showing evidence that numerous specific memory processes may be disrupted, including learning/acquisition, storage/consolidation, and retrieval . Anywhere from 20 to 40% of HIV+ individuals exhibit a primary retrieval profile of memory deficits consistent with injury to fronto-striato-thalamo-cortical circuits. This retrieval profile is characterized by difficulties in bringing previously stored information into conscious awareness and is evidenced by deficits in free recall of information that are ameliorated when structured retrieval cues are provided . A retrieval profile, which is sometimes referred to as a mixed encoding/retrieval profile, can suggest that an individual only partially encodes the target information. This is because stimuli that were processed only in part are difficult to spontaneously recall in their entirety, but may be accurately recognized when presented.

HIV-associated deficits in learning and memory have been linked to poorer everyday functioning, including medication management skills and cART non-adherence. Deficits in acquisition and delayed free recall as a global composite index have been consistently associated with poor performance on laboratory-based medication management tasks across varying types of stimuli . Among the two studies that we found examining specific memory components in the context of adherence, measures of delayed free recall were more consistently associated with medication adherence , whereas indices of initial learning show more variable associations. Yet the specificity of such delayed recall deficits is difficult to determine, as they may be a consequence of problems with encoding, forgetting, and/or retrieval. Thus, while learning and memory are consistently associated with medication management in the laboratory and daily life, little is known about the specific profile that may be driving these relationships. Identification of such profiles at the levels of both group data and individual participants is important in order to enhance the clinical identification of persons at risk for non-adherence and develop tailored compensatory mnemonic approaches to improve adherence. To that end, Wright and colleagues applied the item specific deficit approach , an item analytic method, to study the association between cART adherence and word list encoding, consolidation, and retrieval as measured by the California Verbal Learning Test in 75 HIV+ participants. The ISDA indices were developed as a novel method for categorizing encoding, consolidation and retrieval deficits. The ISDA encoding index is constructed by summing the number of items recalled in less than three of the five learning trials, the ISDA consolidation index reflects the number of items recalled during the learning trials but not recalled again during any of the recall trials , and retrieval deficits are indexed by the number of learned items recalled inconsistently across short- and long-delay trials. To control for potential group differences in learning, the consolidation and retrieval indices are each divided by the total words recalled at least once during the learning trials. When using the ISDA Wright and colleagues found that, compared to healthy adults, HIV+ individuals demonstrated poorer performance on the CVLT regardless of their level of antiretroviral adherence. Additionally, while both HIV+ groups demonstrated similar ISDA encoding deficits, only non-adherers demonstrated ISDA retrieval deficits compared to HIV− participants. Additionally, retrieval abilities, as measured by the ISDA indices, accounted for a greater proportion of variance in long-delay free recall performance for poor adherers than for good adherers. While the study conducted by Wright and colleagues provided initial evidence of an association between retrieval deficits and sub-optimal adherence in HIV, no studies have evaluated the association between sub-optimal adherence and traditional list learning profiles that also incorporate retention and recognition performance as is commonly done in clinical research and practice. At the group level, such profile-based approaches have a long tradition of utility in distinguishing between different neuropsychological disorders. For example, retrieval deficits in the context of significantly improved recognition are commonly associated with “subcortical” dementias . “Cortical” dementias , on the other hand, are sometimes differentiated by rapid forgetting, minimal improvement on recognition, and high rates of cued recall intrusions . Our approach here is to employ those same cognitive psychology approaches of learning and memory profile distinctions to better understand the cognitive architecture of non-adherence. To further enhance the clinical relevance of this group-level analytic approach, we also propose to classify individuals’ profiles as reflective of problems with encoding versus retrieval using an established data-based algorithm that has shown utility differentiating traditionally cortical versus sub-cortical diseases . This algorithm-based individual profile approach has proven to be a worthwhile complement to group-based analyses by allowing for clinically-relevant classification accuracy data and demonstrating the heterogeneity of profiles within these traditionally “cortical” and “sub-cortical” groups.