For instance, HIV-1 Tat protein is produced in infected astrocytes and may be secreted and taken up by neighbouring cells, and has been identified as a potential factor in the pathophysiology of HAND. Expression of the CCL2 gene has been shown to be directly transactivated by HIV-1 Tat protein in human astrocytes. Although we cannot determine from this study whether the CCL2 gene is solely driving the CCL2 gradient or whether the presence of infected cells or HIV- 1 Tat are also contributing to CCL2 expression, the results suggest a link between CCL2 genotype and cognition that warrants further study. Overall, these results underscore the importance of examining intermediate phenotypes as modulating factors that may link host genotype to cognitive outcomes in HIV.These compounds have benefited human society since antiquity, applied as materials , traditional medicines , pharmaceuticals , and cosmetics . The versatile functions and applications of terpenes owe to their diverse chemical structures, with more than 55,000 molecules known . Nature employs a concise paradigm to build such structures, in which isopentenyl pyrophosphate and dimethylallyl pyrophosphate , fivecarbon units derived from the 2-C-methyl-D-erythritol 4-phosphate pathway or mevalonate pathway , are first polymerized head-to-tail into prenyl pyrophosphates, including geranyl , farnesyl , and geranylgeranyl pyrophosphates. Then, the prenyl pyrophosphates are processed, mostly cyclized, by terpene synthases to produce terpene scaffolds . Lastly, the optional tailoring proteins, including P450s and glycosyltransferases , modify the scaffolds to afford the final products. Through this paradigm,greenhouse growing racks structural diversities of the final products are introduced from different numbers of five-carbon units polymerized, varied cyclizations , and optional post modifications.

The final products are usually with multiple of five carbons. Terpenes with non-multiple of five carbons are rare in nature and challenging to sample, even though this chemical space is valuable to explore for novel applications. We reported heterologous expression of the lepidopteran mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel sesquiterpene analogs containing 16 carbons . The LMVA pathway produces 6-carbon analogs of IPP and DMAPP, homoIPP and homo-DMAPP , in a reaction sequence highly similar to the canonical MVA pathway . The difference is that the thiolase condenses a propionyl-CoA and an acetyl-CoA making 3-ketovaleryl-CoA in the LMVA pathway instead of the condensing two acetyl-CoAto produce acetoacetyl-CoA in the canonical MVA pathway . The “extra” carbon from the propionyl-CoA is transformed into HIPP/HDMAPP, combined with two 5-carbon isoprenoid precursors to afford the sixteen-carbon products. With the LMVA pathway expressed in E. coli, we established a biosynthesis platform for novel homoterpenes deviating from the “multiple of five carbons” rule. While the previous study successfully produced the final homosesquiterpenes, the details of the LMVA pathway, especially the production of HIPP and HDMAPP in the E. coli host was not assayed. Moreover, the production needs optimization because low C16 terpene titers have hindered us from accumulating and purifying these products for characterization. Here, we investigate the LMVA pathway by introducing a promiscuous phosphatase, NudB , to hydrolyze the terpene precursors to their corresponding alcohols. These alcohols are readily detected by gas chromatography . Using the alcohols as the final product, we were able to engineer and optimize the LMVA pathway to increase the production of HIPP, the direct product of LMVA and the starting substrate for homoterpenes synthesis. Also, the higher 3-methyl-3-buten-1-ol analogs have excellent fuel properties, making them candidates for next-generation bio-fuels.All the plasmids and primers were designed using j5 DNA assembly unless otherwise indicated, and listed in Table S1 and Table S2, respectively. The plasmids are publicly available through the Joint Bio-Energy Institute registry . Primers were purchased from Integrated DNA Technologies .

PCR amplifications were performed on an Applied Biosystems Veriti Thermal Cycler using Phusion™ High-Fidelity DNA Polymerase or PrimeSTAR Max DNA Polymerase . Gene codon optimization was conducted using the IDT Codon Optimization Tool . Gene fragments were synthesized by Integrated DNA Technologies . Plasmid isolation was carried out using QIAprep Spin Miniprep Kit or by the plasmid DNA preparation service provided by Genewiz, South San Francisco, CA. DNA gel extractions were performed using Zymoclean Gel DNA recovery Kit . Sanger sequencing of plasmids and bacterial clones was supplied by Genewiz, South San Francisco, CA. The plasmid sequences were verified using the DIVA sequence validation service performed by the Synthetic Biology Informatics Group of the Joint BioEnergy Institute . DNA gel photos were taken using BioSpectrum Imaging System . The atoB knockout and atoB yqeF double knockout strains were generated using the λ red recombinase protocol previously described . A kanamycin resistance cassette flanked by FLP recognition target sites was amplified from the plasmid pKD13 with primers KO_kan_F and KO_kan_R.The homology regions were designed to knock out the entire gene except for the starting ATG and the last 21 base pairs to avoid disrupting potential downstream ribosomal binding sites. The upstream and downstream homology sequences were combined with the kanamycin cassette by using NEBuilder HiFi DNA Assembly , and a second PCR was done to amplify the three-part fusion DNA sequences using primer pairs atoB_US_F/atoB_DS_R or yqeF_US_F/yqeF_DS_R using the corresponding assembly product. To generate marker-free atoB knockout, E. coli 6C01, the pKD46 plasmid with the λ red recombination genes and a temperature-sensitive replicon was transformed into chemically competent E. coli BL21 cells and selected on carbenicillin LB agar plates at 30oC. Plasmid-containing cells were made electrocompetent, and expression of recombination genes was induced with 0.1% arabinose. ~600 ng of the three-part PCR product was introduced into the cells by electroporation, and positive colonies were selected for on LB kanamycin plates grown at 37oC overnight.

To remove the kanamycin resistance cassette, pCP20 was transformed into the selected kanamycin resistance cells using electroporation and selected on carbenicillin and kanamycin LB agar plates at 30oC. The resulting colonies were streaked on an LB plate without antibiotic and grew overnight at 42oC to remove the cassette. The resulting colonies were streaked on an LB plate without antibiotic, a carbenicillin LB agar plate, and a kanamycin LB agar plate, respectively, to confirm the loss of the kanamycin cassette and pCP20. Finally, the knockout of atoB was confirmed via colony PCR using the primer pair atoB_C_F/atoB_C_R.To generate maker-free atoB yqeF double knockout, E. coli 6C02, the same protocol for atoB knockout was employed. The three-part PCR product was introduced into 6C01. The knockout of yqeF was confirmed via colony PCR using the primer pair yqeF_C_F/yqeF_C_R. Two plasmids bearing the pathway genes were co-transformed into E. coli expression hosts using a room temperature electroporation method,vertical hydroponic garden in which the electrocompetent bacterial cells were prepared at room temperature freshly . The transformed cells were plated on LB agar plates added with carbenicillin and chloramphenicol, and the resulting plates were incubated at 30oC until colonies appeared.The grown cultures were inoculated into 10 mL production media in 25 mL glass tubes, and the cultures were grown at 37oC, 200 rpm until OD600 reached 0.8. After incubating on ice for 10 min, isopropyl-β-D-thiogalactopyranoside and substrates were added to the cultures, respectively. The cultures were incubated at 18oC, 200 rpm for 72 h to produce isoprenol analogs. After the production, a ten-fold dilution of the production broth was subjected to endpoint optical density measurement using the cuvette port of a SpectraMax M2e plate reader . All the production runs were conducted in biological triplicate. GC-MS analysis was performed on an Agilent Intuvo 9000 system equipped with the pneumatic switching device using two tandem DB-WAX columns with a helium flow of 1 mL/min at the first column and 1.2 mL/min at the second column. The inlet temperature, the Intuvo flow path temperature, and the MS transfer line temperature are 250oC. 1 μL sample was injected using splitless mode. The oven starting temperature was 60oC held for 2 min. The temperature increased at 15oC per minute until 120oC. Then the temperature was increased at 30oC per minute until 245oC. After the temperature ramping program, a post-run was conducted at 250oC for 5 min with a helium flow of -2.691 mL/min at the first column and 3.106 mL/min at the second column.

The homosesquiterpene biosynthesis pathway we constructed in E. coli contains two sections to incorporate propionate into C16H26 terpenes. In the first section, the LMVA pathway transforms propionate into HIPP and HDMAPP. In the second section, these C6 building blocks are transformed into terpenes . The production of those C16H26 terpenes was previously confirmed using GC-MS and GC-MS-TOF analysis and 13C labeling experiments. However, several challenges hindered us from purifying the final products for detailed structural characterization via nuclear magnetic resonance : 1) the C16H26 terpenes were produced at low titers, 1.05 mg/L in the best case and 2) the ratio of C16 terpenes to total terpenes was modest, 11.25% in the best scenario. Moreover, this pathway was expected to produce higher terpenes with molecular formulas of C17H28 and C18H30, but we did not detect these terpenes. We suspected the low production and low ratio of the homoterpenes may be ascribed to insufficient production of the C6 isoprenoid precursors, HIPP and HDMAPP. To assess the production of the C6 isoprenoid precursors, we tested the production of their corresponding alcohols in an E. coli strain co-expressing the LMVA pathway and NudB. NudB is a phosphatase and works with another endogenous phosphatase to dephosphorylate C5-C15 prenyl diphosphates into their corresponding alcohols in E. coli . To construct the production stain, we transformed pNudB and pJH10 into E. coli BAP1 . The resulting strain was grown and induced for production, with sodium propionate added at the time of induction. After production, we extracted the production broth using ethyl acetate, and the organic phase was analyzed by GC-FID and GC-MS. In the analysis, GC-FID detected a peak in the production broth, and this peak was absent in the negative control .This peak also has the same retention time as the C6-isoprenol standard . GC-MS indicated the mass spectrum of the new peak is similar to the spectrum of the C6-isoprenol standard, with major peaks at m/z = 67, 55, 82, indicating the new peak has similar electron ionization induced fragmentation to C6-isoprenol . GC analysis confirmed the production of C6-isoprenol by the production strain, suggesting HIPP was produced. To evaluate the production level of HIPP, we quantified the C6-isoprenol at 5.5 mg/L using the C6-isoprenol standard curve . In the GC analysis, we also noticed the production of isoprenol , the hydrolyzed product of IPP, at 4.1 mg/L , indicating the production strain also produces a comparable amount of IPP as HIPP. We reasoned that IPP might come from the E. coli endogenous MEP pathway and the promiscuity of the LMVA pathway, in which the thiolase accepted two molecules of acetyl-CoA. We also attempted to confirm the production of -C6-prenol, the proposed hydrolyzed product of HDMAPP, using a synthesized standard with GC-FID and GC-MS. However, -C6-prenol was not detected in the production broth, suggesting the idi gene, encoding the isopentenyl-diphosphate delta-isomerase, might not function normally to transform HIPP to HDMAPP . The results of these production runs indicated the supplement of the C6 isoprenoid precursors from the LMVA pathway is at a low level, comparable to the supply of the normal C5 isoprenoid precursors. To address the insufficient supplement of HIPP, we revisited the LMVA pathway for C6-isoprenol production in E. coli. Like the natural MVA pathways, the previous work employed a thiolase to condense propionyl-CoA and acetyl-CoA to afford 3-ketovaleryl-CoA . Because the predicted thiolase from Bombyx mori does not express in E. coli, we used PhaA from Acinetobacter strain RA3849. PhaA has been well characterized in PHA/PHB production, in which PhaA accepts propionyl-CoA and acetyl-CoA. However, PhaA also converts two acetyl-CoA to acetoacetylCoA , an intermediate for IPP production via the thiolase LMVA pathway. Moreover, a previous study has shown that PhaA homologs catalyze the degradation reaction better than the condensation reaction , suggesting that the degradation of 3-ketovaleryl-CoA is a favored direction in the PhaA-catalyzed reversible reaction. Hence, we suspected the thiolase catalyzed reaction is the bottleneck in the thiolase LMVA pathway for HIPP production.