Heterologous Gene Expression of N-Terminally Truncated Variants of LipPks1 Suggests a Functionally Critical Structural Motif in the N-terminus of Modular Polyketide Synthase

Abstract

Streptomyces genomes have a high G + C content and typically use an ATG or GTG codon to initiate protein synthesis. Although gene-finding tools perform well in low GC genomes, it is known that the accuracy in predicting a translational start site (TSS) is much less for high GC genomes. LipPks1 is a Streptomyces-derived, well-characterized modular polyketide synthase (PKS). Using this enzyme as a model, we experimentally investigated the effects of alternative TSSs using a heterologous host, Streptomyces venezuelae. One of the TSSs employed boosted the protein level by 59-fold and the product yield by 23-fold compared to the originally annotated start codon. Interestingly, a structural model of the PKS indicated the presence of a structural motif in the N-terminus, which may explain the observed different protein levels together with a proline and arginine-rich sequence that may inhibit translational initiation. This structure was also found in six other modular PKSs that utilize noncarboxylated starter substrates, which may guide the selection of optimal TSSs in conjunction with start-codon prediction software.

Short-chain ketone production by engineered polyketide synthases in Streptomyces albus

“…Gene-finding tools perform well in low GC content genomes but are much less accurate for high GC genomes such as Streptomyces genomes 34 . In fact, we found a TSS that greatly outperforms the originally annotated start codon in heterologous expression of LipPks1 20 . Because most bioactive PKS products originate from Strepromyces , more accurate gene-finding tools would facilitate heterologous expression of the PKSs.…”

“…Recently, we found that the employment of an alternative translational start site (TSS) in LipPks1 dramatically increased the protein level of LipPks1 + TE and the corresponding product levels, 3-hydroxy acids, in a heterologous Streptomyces host, Streptomyces venezuelae ATCC 10712 (hereafter referred to S . venezuelae ), compared to the originally annotated start codon 20 . Using the newly identified TSS, which truncates a part of the N -terminal tail of LipPks1, we redesigned the ketone PKS genes (Fig.…”

“…Similarly, P37 was constructed using a PKS gene generated with NdeI and EcoRI from pSY075 17 . pSY172 encodes N -terminal tail-truncated LipPks1 + TE 20 . The PKS gene was cut with NdeI and EcoRI and inserted into p21 (pre-pSY186).…”

Technical Advances to Accelerate Modular Type I Polyketide Synthase Engineering towards a Retro-biosynthetic Platform

“…A major consideration in the production stage is the selection of a growth medium. A wellselected medium facilitates the strain's growth and PKS biosynthesis [62,63] and ultimately increases the chance of detecting the final product [64], considering most engineered PKSs result in a decreased production [6,8]. The media preferences for the PKS production vary from host to host.…”

A bimodular PKS platform that expands the biological design space

“…Previously, our group has engineered three major PKS elements in the first module of lipomycin: 1) an inserted TE to produce 3-hydroxy acids (Yuzawa et al, 2017a), 2) a KR knockout and AT domain swap to produce short chain ketones (Yuzawa et al, 2018a(Yuzawa et al, , 2017b, and 3) reductive loop (RL) exchanges to produce saturated, short chain carboxylic acids (Zargar et al, 2019). The design space expands considerably with multiple module systems, and in this work, we build on our single module platform by combining multiple PKS manipulations (KR knockouts, reductive loop (RL) swaps, AT swaps, fused TE) in a biomodular system to produce novel biomolecules, namely biofuels and specialty chemicals.…”