Synthetic genomes unveil the effects of synonymous recoding.

Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers1-4. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement-recoding-is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a synthetic Escherichia coli genome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organism's genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.

A swapped genetic code prevents viral infections and gene transfer.

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer1-6. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus7-9, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature10. Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.

Genetic engineering of the precursor supply pathway for the overproduction of the nC14-surfactin isoform with promising MEOR applications.

BACKGROUND: Surfactin, a representative biosurfactant of lipopeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a ß-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. RESULTS: In this study, the fatty acid precursor supply pathway in Bacillus subtilis 168 for surfactin production was strengthened through two steps. Firstly, pathways competing for the precursors were eliminated with inactivation of pps and pks. Secondly, the plant medium-chain acyl-carrier protein (ACP) thioesterase (BTE) from Umbellularia californica was overexpressed. As a result, the surfactin titer after 24 h of cultivation improved by 34%, and the production rate increased from 0.112 to 0.177 g/L/h. The isoforms identified by RP-HPLC and GC-MS showed that the proportion of nC14-surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC14-surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC14-surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. CONCLUSION: The obtained results identified that enhancing the supply of fatty acid precursor is very essential for the synthesis of surfactin. At the same time, this study also proved that thioesterase BTE can promote the production of nC14-surfactin and experimentally demonstrated its higher surface activity and better performance in oil-washing. These results are of great significance for the MEOR application of surfactin.

Acetoin modulates conformational change of surfactin: Interfacial assembly and crude oil-washing performance.

Due to its special structure, the cyclic lipopeptide surfactin showed remarkable responsiveness to stimuli such as pH, temperature and metal ions. However, few studies investigated the effect of fermented by-products on the conformational change and interfacial assembly of surfactin. Here, the effect of acetoin, a primary metabolite of Bacillus subtilis, on the conformational change and interfacial assembly of surfactin was studied in detail. Surface tension measurements showed that the critical micelle concentration (CMC) of surfactin increased from 1.14 × 10-5 to 4.32 × 10-5 M in the presence of acetoin. Moreover, acetoin has increased the interfacial tension of surfactin aqueous solution-crude oil from 1.08 mN/m to 3.01 mN/m. Circular dichroism (CD) spectra and dynamic light-scattering (DLS) further demonstrated that acetoin had induced the conformational transition of surfactin from ß-sheet to ß-turn structure, and caused surfactin forming some larger micelle aggregations. Afterwards, it was further found that acetoin decreased the oil sand cleaning efficiency of surfactin from 59.7% to 6.6%, and deteriorated the O/W emulsion stability and altered the silicate wettability toward less water wet state. Based on the experimental results, a possible mechanism of the interaction between surfactin and acetoin was proposed.

Efficient production of surfactin from xylose-rich corncob hydrolysate using genetically modified Bacillus subtilis 168.

As one of the most powerful biosurfactants, surfactin has extensive application prospects in numerous industrial fields. Bacillus subtilis 168 was genetically modified to produce surfactin by increasing the supply of the precursor fatty acyl-CoA by overexpressing 4' phosphopantetheinyl transferase, medium-chain acyl-acyl carrier protein (ACP) thioesterase and fatty acyl CoA ligase (encoded by sfp, bte, and yhfL, respectively), and knocking out acyl-CoA dehydrogenase (encoded by fadE). The resulting recombinant strain BSFX022 produced 2203 mg/L surfactin with xylose as carbon source. The lower accumulation of organic acids with xylose as carbon source made it possible to maintain surfactin production in a non-buffered fermentation system, and the yield reached 2074 mg/L. Furthermore, to reduce the costs, waste biomass such as corncob hydrolysate and monosodium glutamate wastewater (MGW) were used, and 2032 mg/L of surfactin was produced in the optimal waste-based medium. To our best knowledge, this is the first report of surfactin production using genetically modified Bacillus subtilis 168 with xylose as carbon source.

Rational strain improvement for surfactin production: enhancing the yield and generating novel structures.

Surfactin, one of the most powerful microbial surfactants, is a lipopeptide-type biosurfactant which combines interesting physicochemical properties and biological activities. However, the high cost caused by its low productivity largely limits the commercial application of surfactin. Hence, many engineered bacterium have also been used to enhance surfactin biosynthesis. This review briefly summarizes the mechanism of surfactin biosynthesis, highlighting the synthesis pathway of N-terminally attached fatty acids, and outlines the main genetic engineering strategies for improving the yield and generating novel structures of surfactin, including promoter engineering, enhancing efflux systems, modifying the transcriptional regulatory genes of surfactin synthase (srfA), genomics and transcriptomics analysis, non ribosomal peptide synthetase (NRPS) domain and combinatorial biosynthesis. Finally, we discuss the future prospects of the research on surfactin.

Genome and transcriptome analysis of Bacillus velezensis BS-37, an efficient surfactin producer from glycerol, in response to d-/l-leucine.

Surfactin is one of the most widely studied biosurfactants due to its many potential applications in different fields. In the present study, Bacillus velezensis BS-37, initially identified as a strain of Bacillus subtilis, was used to efficiently produce surfactin with the addition of glycerol, an inexpensive by-product of biodiesel production. After 36 hr of growth in glycerol medium, the total surfactin concentration reached more than 1,000 mg/L, which was two times higher than that in sucrose medium. Moreover, the addition of l- and d-Leu to the culture medium had opposite effects on surfactin production by BS-37. While surfactin production increased significantly to nearly 2,000 mg/L with the addition of 10 mM l-Leu, it was dramatically reduced to about 250 mg/L with the addition of 10 mM d-Leu. To systemically elucidate the mechanisms influencing the efficiency of this biosynthesis process, we sequenced the genome of BS-37 and analyzed changes of the transcriptome in glycerol medium in response to d-/l-leucine. The RPKM analysis of the transcriptome of BS-37 showed that the transcription levels of genes encoding modular surfactin synthase, the glycerol utilization pathway, and branched-chain amino acid (BCAA) synthesis pathways were all at a relatively high level, which may offered an explanation why this strain can efficiently use glycerol to produce surfactin with a high yield. Neither l-Leu nor d-Leu had a significant effect on the expression of genes in these pathways, indicating that l-Leu plays an important role as a precursor or substrate involved in surfactin production, while d-Leu appears to act as a competitive inhibitor. The results of the present study provide new insights into the synthesis of surfactin and ways of its regulation, and enrich the genomic and transcriptomic resources available for the construction of high-producing strains.